A major problem when xylose is used for ethanol production is the intercellular redox imbalance a... more A major problem when xylose is used for ethanol production is the intercellular redox imbalance arising from different coenzyme specificities of xylose reductase (XR) and xylitol dehydrogenase. The residue Lys21 in XR from Pichia stipitis was subjected to site-directed mutagenesis to alter its coenzyme specificity. The N272D mutant exhibited improved catalytic efficiency when NADH was the coenzyme. Both K21A and K21A/N272D preferred NADH to NADPH, their catalytic efficiencies for NADPH were almost zero. The catalytic efficiency of K21A/N272D for NADH was almost 9-fold and 2-fold that of K21A and the wild-type enzyme, respectively. Complete reversal of coenzyme specificity toward NADH and improved catalytic efficiency were achieved.
Zhong Nan Da Xue Xue Bao Yi Xue Ban Journal of Central South University Medical Sciences, Apr 1, 2007
OBJECTIVE: To detect two exons of Duchenne muscular dystrophy (DMD) gene and a gender discriminat... more OBJECTIVE: To detect two exons of Duchenne muscular dystrophy (DMD) gene and a gender discrimination locus amelogenin gene by single cell triplex PCR, and to evaluate the possibility of this technique for preimplantation genetic diagnosis (PGD) in DMD family with DMD deletion mutation.METHODS: Single lymphocytes from a normal male, a normal female, two DMD patients (exon 8 and 47 deleted, respectively) and single blastomeres from the couples treated by the in vitro fertilization pre-embryo transfer (IVF-ET) and without family history of DMD were obtained. Exons 8 and 47 of DMD gene were amplified by a triplex PCR assay, the amelogenin gene on X and Y chromosomes were co-amplified to analyze the correlation between embryo gender and deletion status.RESULTS: In the normal single lymphocytes, the amplification rate of exons 8 and 47 of DMD and amelogenin gene were 93.8%, 93.8%, and 95.3% respectively. The false positive rate was 3.3%. In the exon 8 deleted DMD patient, the amplification rate of exon 47 of DMD and amelogenin gene was 95.8%, and the false positive rate was 3.3%. In the exon 47 deleted DMD patient, the amplification rate of exon 8 of DMD and amelogenin gene was 95.8%, and the false positive rate was 0. In the single blastomeres, the amplification rate of exons 8 and 47 of DMD and amelogenin gene was 82.5%, 80.0% and 77.5%, respectively, and the false positive rate was 0.CONCLUSION: The single cell triplex PCR protocol for the detection of DMD and amelogenin gene is highly sensitive, specific and reliable, and can be used for PGD in those DMD families with DMD deletion mutation.
Zhonghua Yan Ke Za Zhi Chinese Journal of Ophthalmology, Aug 1, 2005
To evaluate the validity of confocal microscopy in estimating curative effect and in directing th... more To evaluate the validity of confocal microscopy in estimating curative effect and in directing the treatment for fungal keratitis in the process of antifungal chemotherapy. Fifty-eight patients, who were confirmed fungal infection by confocal microscopy, were selected from 328 patients with fungal keratitis. All patients received routine topical and/or oral antifungal medication, and were examined by confocal microscopy once a week and one week after discontinuation of the treatment. The density of hyphae in the corneal lesion, the configuration of inflammatory cells and keratocyte were recorded. Antifungal chemotherapy was adjusted according to examination results and medicines were changed accordingly. If no hyphae were detected by confocal microscopy, antifungal medication was maintained for one week and then discontinued. All patients were followed up for two months to ensure no relapse of fungal infection. Fifty three patients were cured. The area of corneal lesions began to reduce 7 days after the beginning of antifungal chemotherapy. Confocal microscopy examination revealed that the hypha positive sites and the density of hypha were reduced gradually; inflammatory cells also decreased, the configuration of corneal lesion was transformed from asymmetry to symmetry; and normal keratocytes could be detected gradually. After 14 days of treatment, ulcers healed up in 37 cases and no hyphae and inflammatory cells were found in 23 cases. After 28 days of treatment, all corneal ulcers healed up; hyphae and inflammatory cells were completely disappeared in 31 patients, but a few hyphae still could be found in 22 patients. Antifungal chemotherapy was tapered gradually if no hyphae and inflammatory cells were detected by confocal microscopy. There was no relapse of fungus infection during 2-month follow-up. Infection deteriorated in the other five patients within 7 days, which showed increased density of hypha and inflammatory cells under confocal microscopy examination. All of them were treated with a penetrating keratoplasty to save the eyeball. Confocal microscopy is an ideal method for the evaluation of curative effects of fungal keratitis in the process of antifungal chemotherapy. This is also a valuable objective tool in directing antifungal medication.
Recently, we have described a novel gene, DD3, which is one of the most prostate cancer-specific ... more Recently, we have described a novel gene, DD3, which is one of the most prostate cancer-specific genes described to date (Bussemakers, M. J. G., van Bokhoven, A., Verhaegh, G. W., Smit, F. P., Karthaus, H. F. M., Schalken, J. A., Debruyne, F. M. J., Ru, N., and Isaacs, W. B. (1999) Cancer Res. 59, 5975-5979). The prostate cancer-specific expression of DD3 indicates that the DD3 gene promoter is a promising tool for the treatment of prostate cancer. To identify the promoter elements that are responsible for the prostate cancer-specific expression of DD3, we have isolated and characterized the DD3 promoter. Sequence analysis of the DD3 5'-flanking region was performed and several promoter-human growth hormone reporter constructs were prepared, which were transiently transfected in the DD3-positive cell line LNCaP and several DD3-negative cell lines. Using a 500-base pair DD3 promoter construct, we could detect promoter activity in LNCaP cells, which was not affected by increasing the size of the constructs. Truncated constructs, however, showed an increased transcriptional activity, suggesting the presence of a silencer that negatively regulates the expression of DD3. DNase-I footprint analysis, using nuclear extracts from LNCaP cells, revealed the presence of three DNase-I-protected areas within the DD3 proximal promoter. We show that the high mobility group I(Y) protein binds to one of the DNase-I-protected areas and recruits another, yet unidentified, protein to the DD3 promoter in LNCaP cells.
In the title compound, [Co(C(13)H(16)NOSe(2))(2)]·CH(3)CN, the Co(II) atom is four-coordinated by... more In the title compound, [Co(C(13)H(16)NOSe(2))(2)]·CH(3)CN, the Co(II) atom is four-coordinated by two N,O-bidentate Schiff base ligands, resulting in a distorted tetra-hedral coordination for the metal ion.
The Chinese-German Journal of Clinical Oncology, 2008
ABSTRACT ObjectiveTo investigate the mechanism of apoptosis of myelocytic leukemia lines NB4, K56... more ABSTRACT ObjectiveTo investigate the mechanism of apoptosis of myelocytic leukemia lines NB4, K562 and THP-1 cells induced by curcumin. MethodsAfter the cells were treated with curcumin at different concentration (25, 12.5, 6.25, 3.125, 0 μmol/L) for various times (0, 12, 24, 48 h), flow cytometry (FCM) was used to determine the rate of apoptosis of cells. After the cells were treated with curcumin at 25 μmol/L for 24 h, flow cytometry was used to determine the expression level of Fas, and Western blot was performed to determine the expression of Caspase-8 and Caspase-9. Results(1) Curcumin could induced the apoptosis of NB4, K562 and THP-1 cells in a time-and dose-dependent manner. After the cells were treated with curcumin at 25 μmol/L for 48 h, the rate of apoptosis of cells was over fifty percent. (2) Fas level showed remarkable increase (P < 0.01) after above cells were treated with 25 μmol/L curcumin for 24 h. (3) The apoptosis proteins of Caspase-8 and Caspase-9 were also increased obviously (P < 0.01) after the cells were treated with 25 μmol/L curcumin for 24 h. ConclusionThe molecular pathway of apoptosis of myelocytic leukemia lines induced by curcumin are concerned with death receptor and mitochondria.
The phenolic profiles and antioxidant activity of litchi pulp of 13 varieties were investigated. ... more The phenolic profiles and antioxidant activity of litchi pulp of 13 varieties were investigated. The free, bound and total phenolic contents were 66.17-226.03, 11.18-40.54, and 101.51-259.18 mg of gallic acid equivalents/100 g, respectively. The free, bound and total flavonoid contents were 16.68-110.33, 10.48-22.75, and 39.43-129.86 mg of catechin equivalents/100 g, respectively. Free phenolics and flavonoids contributed averagely 80.1% and 75% to their total contents, respectively. Six individual phenolics (gallic acid, chlorogenic acid, (+)-catechin, caffeic acid, (-)-epicatechin, and rutin) were detected in litchi pulp by HPLC. The contents of each compound in free and bound fractions were determined. Significant varietal discrepancy in antioxidant activity was also found by FRAP and DPPH scavenging capacity methods. Antioxidant activity was significantly correlated with phenolic and flavonoid contents. Thus, phenolics and flavonoids exist mainly in the free form in litchi pulp. There were significant varietal differences in phytochemical contents and antioxidant activity of litchi pulp.
A 2-aminothiazole derivative 1 was developed as a potential inhibitor of the oncology target AKT,... more A 2-aminothiazole derivative 1 was developed as a potential inhibitor of the oncology target AKT, a serine/threonine kinase. When incubated in rat and human liver microsomes in the presence of NADPH, 1 underwent significant metabolic activation on its 2-aminothiazole ring, leading to substantial covalent protein binding. Upon addition of glutathione, covalent binding was reduced significantly, and multiple glutathione adducts were detected. Novel metabolites from the in vitro incubates were characterized by LC-MS and NMR to discern the mechanism of bioactivation. An in silico model was developed based on the proposed mechanism and was employed to predict bioactivation in 23 structural analogues. The predictions were confirmed empirically for the bioactivation liability, in vitro, by LC-MS methods screening for glutathione incorporation. New compounds were identified with a low propensity for bioactivation.
A major problem when xylose is used for ethanol production is the intercellular redox imbalance a... more A major problem when xylose is used for ethanol production is the intercellular redox imbalance arising from different coenzyme specificities of xylose reductase (XR) and xylitol dehydrogenase. The residue Lys21 in XR from Pichia stipitis was subjected to site-directed mutagenesis to alter its coenzyme specificity. The N272D mutant exhibited improved catalytic efficiency when NADH was the coenzyme. Both K21A and K21A/N272D preferred NADH to NADPH, their catalytic efficiencies for NADPH were almost zero. The catalytic efficiency of K21A/N272D for NADH was almost 9-fold and 2-fold that of K21A and the wild-type enzyme, respectively. Complete reversal of coenzyme specificity toward NADH and improved catalytic efficiency were achieved.
Zhong Nan Da Xue Xue Bao Yi Xue Ban Journal of Central South University Medical Sciences, Apr 1, 2007
OBJECTIVE: To detect two exons of Duchenne muscular dystrophy (DMD) gene and a gender discriminat... more OBJECTIVE: To detect two exons of Duchenne muscular dystrophy (DMD) gene and a gender discrimination locus amelogenin gene by single cell triplex PCR, and to evaluate the possibility of this technique for preimplantation genetic diagnosis (PGD) in DMD family with DMD deletion mutation.METHODS: Single lymphocytes from a normal male, a normal female, two DMD patients (exon 8 and 47 deleted, respectively) and single blastomeres from the couples treated by the in vitro fertilization pre-embryo transfer (IVF-ET) and without family history of DMD were obtained. Exons 8 and 47 of DMD gene were amplified by a triplex PCR assay, the amelogenin gene on X and Y chromosomes were co-amplified to analyze the correlation between embryo gender and deletion status.RESULTS: In the normal single lymphocytes, the amplification rate of exons 8 and 47 of DMD and amelogenin gene were 93.8%, 93.8%, and 95.3% respectively. The false positive rate was 3.3%. In the exon 8 deleted DMD patient, the amplification rate of exon 47 of DMD and amelogenin gene was 95.8%, and the false positive rate was 3.3%. In the exon 47 deleted DMD patient, the amplification rate of exon 8 of DMD and amelogenin gene was 95.8%, and the false positive rate was 0. In the single blastomeres, the amplification rate of exons 8 and 47 of DMD and amelogenin gene was 82.5%, 80.0% and 77.5%, respectively, and the false positive rate was 0.CONCLUSION: The single cell triplex PCR protocol for the detection of DMD and amelogenin gene is highly sensitive, specific and reliable, and can be used for PGD in those DMD families with DMD deletion mutation.
Zhonghua Yan Ke Za Zhi Chinese Journal of Ophthalmology, Aug 1, 2005
To evaluate the validity of confocal microscopy in estimating curative effect and in directing th... more To evaluate the validity of confocal microscopy in estimating curative effect and in directing the treatment for fungal keratitis in the process of antifungal chemotherapy. Fifty-eight patients, who were confirmed fungal infection by confocal microscopy, were selected from 328 patients with fungal keratitis. All patients received routine topical and/or oral antifungal medication, and were examined by confocal microscopy once a week and one week after discontinuation of the treatment. The density of hyphae in the corneal lesion, the configuration of inflammatory cells and keratocyte were recorded. Antifungal chemotherapy was adjusted according to examination results and medicines were changed accordingly. If no hyphae were detected by confocal microscopy, antifungal medication was maintained for one week and then discontinued. All patients were followed up for two months to ensure no relapse of fungal infection. Fifty three patients were cured. The area of corneal lesions began to reduce 7 days after the beginning of antifungal chemotherapy. Confocal microscopy examination revealed that the hypha positive sites and the density of hypha were reduced gradually; inflammatory cells also decreased, the configuration of corneal lesion was transformed from asymmetry to symmetry; and normal keratocytes could be detected gradually. After 14 days of treatment, ulcers healed up in 37 cases and no hyphae and inflammatory cells were found in 23 cases. After 28 days of treatment, all corneal ulcers healed up; hyphae and inflammatory cells were completely disappeared in 31 patients, but a few hyphae still could be found in 22 patients. Antifungal chemotherapy was tapered gradually if no hyphae and inflammatory cells were detected by confocal microscopy. There was no relapse of fungus infection during 2-month follow-up. Infection deteriorated in the other five patients within 7 days, which showed increased density of hypha and inflammatory cells under confocal microscopy examination. All of them were treated with a penetrating keratoplasty to save the eyeball. Confocal microscopy is an ideal method for the evaluation of curative effects of fungal keratitis in the process of antifungal chemotherapy. This is also a valuable objective tool in directing antifungal medication.
Recently, we have described a novel gene, DD3, which is one of the most prostate cancer-specific ... more Recently, we have described a novel gene, DD3, which is one of the most prostate cancer-specific genes described to date (Bussemakers, M. J. G., van Bokhoven, A., Verhaegh, G. W., Smit, F. P., Karthaus, H. F. M., Schalken, J. A., Debruyne, F. M. J., Ru, N., and Isaacs, W. B. (1999) Cancer Res. 59, 5975-5979). The prostate cancer-specific expression of DD3 indicates that the DD3 gene promoter is a promising tool for the treatment of prostate cancer. To identify the promoter elements that are responsible for the prostate cancer-specific expression of DD3, we have isolated and characterized the DD3 promoter. Sequence analysis of the DD3 5'-flanking region was performed and several promoter-human growth hormone reporter constructs were prepared, which were transiently transfected in the DD3-positive cell line LNCaP and several DD3-negative cell lines. Using a 500-base pair DD3 promoter construct, we could detect promoter activity in LNCaP cells, which was not affected by increasing the size of the constructs. Truncated constructs, however, showed an increased transcriptional activity, suggesting the presence of a silencer that negatively regulates the expression of DD3. DNase-I footprint analysis, using nuclear extracts from LNCaP cells, revealed the presence of three DNase-I-protected areas within the DD3 proximal promoter. We show that the high mobility group I(Y) protein binds to one of the DNase-I-protected areas and recruits another, yet unidentified, protein to the DD3 promoter in LNCaP cells.
In the title compound, [Co(C(13)H(16)NOSe(2))(2)]·CH(3)CN, the Co(II) atom is four-coordinated by... more In the title compound, [Co(C(13)H(16)NOSe(2))(2)]·CH(3)CN, the Co(II) atom is four-coordinated by two N,O-bidentate Schiff base ligands, resulting in a distorted tetra-hedral coordination for the metal ion.
The Chinese-German Journal of Clinical Oncology, 2008
ABSTRACT ObjectiveTo investigate the mechanism of apoptosis of myelocytic leukemia lines NB4, K56... more ABSTRACT ObjectiveTo investigate the mechanism of apoptosis of myelocytic leukemia lines NB4, K562 and THP-1 cells induced by curcumin. MethodsAfter the cells were treated with curcumin at different concentration (25, 12.5, 6.25, 3.125, 0 μmol/L) for various times (0, 12, 24, 48 h), flow cytometry (FCM) was used to determine the rate of apoptosis of cells. After the cells were treated with curcumin at 25 μmol/L for 24 h, flow cytometry was used to determine the expression level of Fas, and Western blot was performed to determine the expression of Caspase-8 and Caspase-9. Results(1) Curcumin could induced the apoptosis of NB4, K562 and THP-1 cells in a time-and dose-dependent manner. After the cells were treated with curcumin at 25 μmol/L for 48 h, the rate of apoptosis of cells was over fifty percent. (2) Fas level showed remarkable increase (P < 0.01) after above cells were treated with 25 μmol/L curcumin for 24 h. (3) The apoptosis proteins of Caspase-8 and Caspase-9 were also increased obviously (P < 0.01) after the cells were treated with 25 μmol/L curcumin for 24 h. ConclusionThe molecular pathway of apoptosis of myelocytic leukemia lines induced by curcumin are concerned with death receptor and mitochondria.
The phenolic profiles and antioxidant activity of litchi pulp of 13 varieties were investigated. ... more The phenolic profiles and antioxidant activity of litchi pulp of 13 varieties were investigated. The free, bound and total phenolic contents were 66.17-226.03, 11.18-40.54, and 101.51-259.18 mg of gallic acid equivalents/100 g, respectively. The free, bound and total flavonoid contents were 16.68-110.33, 10.48-22.75, and 39.43-129.86 mg of catechin equivalents/100 g, respectively. Free phenolics and flavonoids contributed averagely 80.1% and 75% to their total contents, respectively. Six individual phenolics (gallic acid, chlorogenic acid, (+)-catechin, caffeic acid, (-)-epicatechin, and rutin) were detected in litchi pulp by HPLC. The contents of each compound in free and bound fractions were determined. Significant varietal discrepancy in antioxidant activity was also found by FRAP and DPPH scavenging capacity methods. Antioxidant activity was significantly correlated with phenolic and flavonoid contents. Thus, phenolics and flavonoids exist mainly in the free form in litchi pulp. There were significant varietal differences in phytochemical contents and antioxidant activity of litchi pulp.
A 2-aminothiazole derivative 1 was developed as a potential inhibitor of the oncology target AKT,... more A 2-aminothiazole derivative 1 was developed as a potential inhibitor of the oncology target AKT, a serine/threonine kinase. When incubated in rat and human liver microsomes in the presence of NADPH, 1 underwent significant metabolic activation on its 2-aminothiazole ring, leading to substantial covalent protein binding. Upon addition of glutathione, covalent binding was reduced significantly, and multiple glutathione adducts were detected. Novel metabolites from the in vitro incubates were characterized by LC-MS and NMR to discern the mechanism of bioactivation. An in silico model was developed based on the proposed mechanism and was employed to predict bioactivation in 23 structural analogues. The predictions were confirmed empirically for the bioactivation liability, in vitro, by LC-MS methods screening for glutathione incorporation. New compounds were identified with a low propensity for bioactivation.
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